Neural stem cells (NSCs) are postulated to be relatively primordial, uncommitted cells that exist in the developing and even adult nervous system and are responsible for giving rise to the array of more specialized cells of the mature CNS1xe2x88x9212. They are operationally defined by their ability (a) to differentiate into cells of all neural lineages (neuronsxe2x80x94ideally of multiple subtypes, oligodendroglia, astroglia) in multiple regional and developmental contexts (i.e., be multipotent); (b) to self-renew (i.e., also give rise to new NSCs with similar potential); (c) to populate developing and/or degenerating CNS regions. An unambiguous demonstration of monoclonal derivation is obligatory to the definitionxe2x80x94i.e., a single cell must possess these attributes. With the earliest recognition that rodent neural cells with stem cell properties, propagated in culture, could be reimplanted into mammalian brain where they could reintegrate appropriately and stably express foreign genes13-16, gene therapists and restorative neurobiologists began to speculate how such a phenomenon might be harnessed for therapeutic advantage as well as for understanding developmental mechanisms. These, and the studies which they spawned (reviewed elsewhere2,9xe2x88x9211,17xe2x88x9221) provided hope that the use of NSCsxe2x80x94by virtue of their inherent biologyxe2x80x94might circumvent some of the limitations of presently available graft material and gene transfer vehicles and make feasible a variety of novel therapeutic strategies20xe2x88x9222. Neural cells with stem cell properties have been isolated from the embryonic, neonatal and adult rodent CNS and propagated in vitro by a variety of equally effective and safe meansxe2x80x94both epigenetic (e.g., with mitogens such as epidermal growth factor [EGF] or basic fibroblast growth factor [bFGF]1,5,16,23xe2x88x9227 or with membrane substrates7) and genetic (e.g., with propagating genes such as vmyc or SV40 large T-antigen1,9xe2x88x9215,17xe2x88x9219,28xe2x88x9232). Maintaining such NSCs in a proliferative state in culture does not appear to subvert their ability to respond to normal developmental cues in vivo following transplantationxe2x80x94to withdraw from the cell cycle, interact with host cells, differentiate appropriately9xe2x88x9216,29xe2x88x9233. These extremely plastic cells migrate and differentiate in a temporally and regionally appropriate manner particularly following implantation into germinal zones throughout the brain. They participate in normal development along the murine neuraxis, intermingling non-disruptively with endogenous progenitors, responding similarly to local microenvironmental cues for their phenotypic determination and appropriately differentiating into diverse neuronal and glial cell types. In addition, they can express foreign genes (both reporter genes and therapeutic genes) in vivo9xe2x88x9221.29xe2x88x9232, and are capable of specific neural cell replacement in the setting of absence or degeneration of neurons and/or glia9.11.31.32.
This paper affirms the potential of clones of human NSCs to perform these critical functions in vitro and in vivo. We show them to be multipotent, self-renewing, engraftable, plastic, and migratory; to secrete gene products that can cross-correct a prototypical neurodegenerative genetic enzymatic defect (a necessary precursor for gene therapy of such diseases); to pursue in vivo (following transplantation into various germinal zones) developmental programs appropriate to a given region and stage (even if different from that from which the cells were initially obtained); to be capable of ex vivo genetic manipulationxe2x80x94e.g., retroviral-mediated transduction of a foreign genexe2x80x94and to be capable, following transplantation, of expressing that transgene in vivo in widely disseminated CNS regions (further establishing gene delivery/therapy potential); and to be capable of differentiating towards replacement of specific deficient neuronal populations in a prototypic mouse mutant model of neurodegeneration and impaired development (suggesting a potential for therapeutic CNS cell replacement). Comparison is also made between the capabilities of human NSCs propagated by either epigenetic (with bFGF) or genetic (via a constitutively downregulated vmyc ) means; the findings suggest that these two common means of propagation are equally effective and safe (inferring that investigators may freely choose the technique that best fits their research or clinical demands.)
We present evidence that neural cells with rigorously defined stem cell features, may, indeed, be isolated from the human brain and may emulate the behavior of NSCs in lower mammals. Not only do these observations vouchsafe conservation of certain neurodevelopmental principles to the human CNS , but they suggest that this class of neural cells may ultimately be applied as well to research and clinical problems in the human. Indeed, not only might the actual human NSC clones described in this report serve that function, but our data suggest that other investigators may readily obtain and propagate such cells from other sources of human material through a variety of equally safe and effective methods (both epigenetic and genetic) with the expectation that such cells will fulfill the demands of multiple research and/or therapeutic problems.
The growing interest in the potential of NSCs has been analogous to that in hematopoietic stem cells. This interest derives from the realization that NSCs are not simply a substitute for fetal tissue in transplantation paradigms or simply another vehicle for gene delivery. Their basic biology, at least as revealed through the examination of cells, appears to endow them with a potential that other vehicles for gene therapy and repair may not possess, For example, that they may integrate into neural structures after transplantation may allow for the regulated release of various gene products as well for literal cell replacement. (While presently available gene transfer vectors usually depend on relaying new genetic information through established neural circuits, which may, in fact, have degenerated and require replacement, NSCs may participate in the reconstitution of these pathways.) The replacement of enzymes and of cells may not only be targeted to specific, anatomically circumscribed regions of CNS, but also, if desired, to larger areas of the CNS in a widespread manner by simple implantation into germinal zones. This ability is important because many neurologic diseases are not localized to specific sites as is Parkinson""s disease. Rather their neuropathology is often extensive, multifocal, or even global (e.g., the lesions present in various traumatic, immunologic, infectious, ischemic, genetic, metabolic, or neurodegenerative processes). These are therapeutic challenges conventionally regarded as beyond the purview of neural transplantation. NSCs, therefore, have helped to broaden the paradigmatic scope of transplantation and gene therapy in the CNS. NSCs pass readily and unimpeded through the blood-brain barrier and deliver their foreign gene products immediately, directly, and, if necessary, in a disseminated fashion to the CNS. In addition, NSCs may be responsive to neurodegeneration, shifting their differentiation to compensate for deficient cell types. The biology underlying these properties may not only be of practical value but might illuminate fundamental developmental mechanisms.
To summarize our results, clones of human NSCsxe2x80x94unambiguously affirmed by the presence of a common retroviral insertion site and propagated by either epigenetic or genetic meansxe2x80x94can participate in normal CNS development in vivo and respond to normal microenvironmental cues, including migration from various germinal zones along well-established migratory routes to widely disseminated regions. A single NSC is capable of giving rise to progeny in all 3 fundamental neural lineagesxe2x80x94neurons (of various types), oligodendroglia, and astroglia (hence, multipotency)xe2x80x94as well as giving rise to new NSCs with similar potential (i.e., self-renewal). In vivo, following transplantation into mouse hosts, a given human NSC clone is sufficiently plastic to differentiate into neural cells of region- and developmental stage-appropriate lineages along the length of the neural axis: into neurons where neurogenesis normally persists, and into glia where gliogenesis predominates, emulating patterns well-established for endogenous murine progenitors, with which they intermingle seamlessly. Thus, for example, they will give rise to neurons following migration into the OB at one end of the neuraxis and into granule neurons in the cerebellum at the other, yet also yield astroglia and oligodendroglia, the appropriate cell types born in the postnatal neocortex, subcortical white matter, and striatum. Of additional significance, as might be expected of a true stem cell, we could demonstrate that many of the neuronal types into which these NSCs could differentiate, are born not at the developmental stage from which the cells were initially obtained (e.g. midgestation), but rather at the stage and region of NSC implantation, thus affirming appropriate temporal (in addition to regional) developmental responsiveness.
Interestingly, the most robust differentiation was ultimately not achieved in the culture dish where cells could maintain a more undifferentiated appearance for prolonged periods, but in the transplanted brain, where they rapidly pursued differentiated phenotypes. This conclusion is also supported by the observation that, for the in vitro experiments, triggering astrogliogenesis (the last cell type typically born in the developing brain) required the presence of co-cultured primary CNS cells (presumably recreating the xe2x80x9cmilieuxe2x80x9d) whereas simple implantation of the NSCs into the in vivo environment was sufficient in the transplant experiments.
Such abundant, genetically-homogeneous, manipulatable cells clearly represent a valuable model for studying human NSC biology in vivo and in vitro. In this paper, we have demonstrated that human NSC clones possess the capabilities that might lead one to expect them to be effective in true clinician situations.
We demonstrate the ability of these cells, in their widely disseminated locations (from even a single, simple implantation procedure) to express a retrovirally-tansduced foreign gene (lacZ), providing promise for future therapeutic gene transfer strategies. That gene products delivered by these human NSCs might be expected to cross-correct dysfunctional neural cells of all types was suggested by our experiments demonstrating the successful delivery of an index therapeutic protein (hexosaminidase) to a prototypical model of neural cells deficient (via targeted mutagenesis) in that specific gene product (Tay-Sachs mouse cells). Tay-Sachs brain cells of neuronal, glial, and even immature neuroepithelial progenitor phenotypes could be effectively rescued by the secretory products of these human NSCs and complement them effectively. Once internalized in the target neural cells, this gene product forestalled pathologic GM2 accumulation in the majority of mutated cells. This successful molecular cross-correction taken together with the cellular transplantation and in vivo migration data help establish the feasibility of human NSC-mediated strategies for the treatment of extensive inherited metabolic and other neurogenetic human diseases for which Tay-Sachs is an exemplar.
In summary, NSCs may be propagated by a variety of means (both epigenetic and genetic) that are comparably effective and safe in yielding engraftable, responsive neural cells (and may, in fact, access common final molecular pathways that interact reversibly with cell cycle regulatory proteins ). Therefore, insights gained from studies of NSCs perpetuated by one technique may be legitimately joined with insights derived from studies employing others to help provide a more complete picture of NSC biology. Furthermore, in helping to resolve debate in the NSC literature as to which techniques are most effective for isolating and manipulating NSCs, and doing so with cells of human origin, the door is open for investigators and/or clinicians to pick the propagation technique that best serves the demands of their particular research or clinical problem. These may have significant practical implications. It is interesting that propagating NSCs by genetic means (e.g. a vmyc construct that is constitutively downregulated by normal developmental mechanisms and environmental cues) appears to be among the safest, easiest, most efficacious, reliable, and cost-effective methods to date for many needs.